Postprint of: Current Microbiology 72(4):370-376 (2016) Title: Influence of temperature and copper on Oxalobacteraceae in soil enrichments 1Helena Gaspar, 1Rui Ferreira, 2Juan Miguel Gonzalez, 1Maria Ivone da Clara and 1Margarida Maria Santana 1ICAAM- Instituto de Ciências Agrárias e Ambientais Mediterrânicas, Departamento de Fitotecnia, Universidade de Évora, Núcleo da Mitra, Ap. 94, 7006-554, Évora, Portugal 2Instituto de Recursos Naturales y Agrobiología, IRNAS-CSIC, Av. Reina Mercedes, 10, E-41012, Sevilla, Spain Correspondence to: Margarida M. Santana, PhD ICAAM- Instituto de Ciências Agrárias e Ambientais Mediterrânicas, Departamento de Fitotecnia, Universidade de Évora, Núcleo da Mitra, Ap. 94, 7006-554, Évora, Portugal Tel. + 351 266 760 851; Fax + 351 266 760 822 E-mail: [email protected] Funding Information: This work was funded by FEDER Funds through the Operational Programme for Competitiveness Factors - COMPETE and National Funds through FCT - Foundation for Science and Technology under the Strategic Project PEst-C/AGR/UI0115/2011. 0 Title: Influence of temperature and copper on Oxalobacteraceae in soil enrichments Abstract β-Proteobacteria is one of the most abundant phylum in soils, including autotrophic and heterotrophic ammonium-consumers with relevance in N circulation in soils. The effects of high temperature events and phytosanitary treatments, such as copper amendments, on soil bacterial communities relevant to N-cycling remain to be studied. As an example, South Portugal soils are seasonally exposed to high temperature periods, the temperature at the upper soil layers can reach over 40 ºC. Here, we evaluated the dynamics of mesophilic and thermophilic bacteria from a temperate soil, in particular of heterotrophic β-Proteobacteria, regarding the ammonium equilibrium, as a function of temperature and copper treatment. Soil samples were collected from an olive orchard in southern Portugal. Selective enrichments were performed from samples under different conditions of temperature (30 and 50 ºC) and copper supplementation (100 and 500 µM) in order to mime seasonal variations and phytosanitary treatments. Changes in the microbial communities under these conditions were examined by DGGE (Denaturing Gradient Gel Electrophoresis), a molecular fingerprint technique. At moderate temperature -30 ºC- either without or with copper addition, dominant members were identified as different strains belonging to genus Massilia, a genus of the Oxalobacteraceae (β-Proteobacteria), while at 50 ºC, members of the Brevibacillus genus, phylum Firmicutes were also represented. Ammonium production during bacterial growth at moderate and high temperatures was not affected by copper addition. Results indicate that both copper and temperature selected specific tolerant bacterial strains with consequences for N-cycling in copper treated orchards. Keywords: Soil β-Proteobacteria; ammonium; copper-tolerance; N-cycling 1 INTRODUCTION Soils are complex biological systems, whose characteristics depend on the interaction of abiotic and biotic factors. The biotic component of soils is greatly ruled by the highly diverse microbial communities and their activities. Between 104-106 different microorganisms and about 1010microbes are estimated per gram of soil [3]. These include soil bacteria with at least 32 phylum-level groups [9]. The dominant phyla are Proteobacteria, Acidobacteria and Actinobacteria. The Proteobacteria usually make up about 39 % of the total soil bacterial communities [9]. Proteobacteria comprises important members for nitrogen and sulfur cycling in soils. The aerobic autotrophic ammonia oxidizing bacteria (AAOB) participate in the nitrification process converting ammonium to nitrite, followed by the conversion of nitrite to nitrate by nitrite oxidizing bacteria (NOB). Ammonia oxidation is considered the limiting step for nitrification in soil ecosystems [1]. AAOB includes the chemolitotrophic β-Proteobacteria genera Nitrosomonas, Nitrosospira and Nitrosolobus, and a member of the γ-Proteobacteria (Nitrosococcus oceanus) [5]. Heterotrophic ammonia oxidizing bacteria are, for instance, Pseudomonas sp. (γ-Proteobacteria), Ochrobactrum (α-Proteobacteria) [10] and Alcaligenes faecalis (β-Proteobacteria) [18]. A recent analysis reports a higher nitrogen circulation activity for AAOB rich soils, correlated with ammonia-oxidizing activities of AAOB, which are 103 to 106 times more abundant than those of heterotrophic AOB [13]. However, in organic-reach soils AAOB decrease in number and activity [28], whereas heterotrophic AOB dominate. Crops are usually treated for prevention of microbial diseases, copper being frequently used as a fungicide [19]. Although copper is an important micronutrient because it is a cofactor for numerous enzymes, its excess causes toxicity, which is associated with the destruction of the iron-sulfur centers of enzymes due to the displacement of iron by copper [12]. Copper treatments can selectively influence the dynamics of soil bacterial communities by modifying the ratio of copper-sensitive to copper-resistant microorganisms. Both Gram-positive and Gram-negative copper-resistant bacteria possess proteins for copper efflux (see e.g. [26]) as a defense mechanism against copper toxicity. Copper addition to crops is being extensively used, for instance, in grape and olive orchards in South Portugal. These soils are often under thermic stress and the upper soil layers reach over 40 ºC during summer. In this study, we assessed the effect of copper and temperature on β-Proteobacteria 2 dynamics and ammonium release in soil, so that future agricultural models can be conceived regarding the maintenance of the diversity and activity of this soil bacterial phylum. MATERIALS AND METHODS Sampling and enrichment cultures Sampling was carried out in an olive orchard in Alentejo, Southern Portugal (38°29'54.1"N 7°45'38.5"W). The studied soil showed moderate drainage, low erosion and low topsoil cntent of organic carbon (i.e., 75 %) [14]. This soil has been previously classified as Haplic Luvisol [8] The analyzed field underwent a phytosanitary treatment with Cuprital® (copper oxychloride) by aspersion on tree crowns. (average air temperature 12 ºC, 100 mm precipitation). Analyzed samples consisted of three adjacent soil cores pooled together which were collected aseptically from topsoil, at 3.5 cm to 7.5 cm depth. Soil temperature was approximately 10ºC at the time of collection. Additional samples were collected from areas treated one month (T0) before and at different periods, six months (T6) and twenty months (T20), previous to this sampling. Soil was collected either between the tree crop line (EC) or below tree crowns (DC). Physico-chemical parameters were estimated following standard procedures by the Laboratory of Agricultural Chemistry [11]. Six gram aliquots of the composite sample T20-EC were added, under sterile conditions, to 100 ml flasks with 15 ml of NB (Nutrient Broth) (Oxoid) pH 7.0, or to NB supplemented with CuSO 4, at 100 μM (pH 7.0) and 500 μM (pH 7.0). The cultures were incubated at 30 or 50 ºC, with shaking at 180 rpm for 24 hours. Serial dilutions from these enrichments were made in NB; dilutions 10-6 and 10-8 were plated on NBA (Nutrient Broth with 15 % w/v Agar) at 50° C for 24 hours and colony-forming units/ml for each enrichment were determined. Measurement of ammonium production The quantification of the ammonium followed the spectrophotometric method described by Taylor et al. [27]. A reaction mix with 40 μL of sterile non-inoculated medium served as blank. Samples 3 were analyzed in triplicate. Concentration values were determined using a calibration curve for 0-5 mM NH4Cl solutions. DNA extraction and PCR-DGGE DNA extraction from enrichments was carried out with DNA Isolation kit PowerSoil® (MoBio Laboratories Inc, USA) following the manufacturer’s instructions. Fingerprints of the whole bacterial communities were obtained through PCR amplifications using the primers 341F-GC and 518R [15, 16]. A nested-PCR strategy was adopted to obtain β-Proteobacteria fingerprints. After the first amplification, with primers Beta 359F and Beta 682R, the second step of the nested-PCR used the primer pair 518F-GC and Beta 682 R (Table 1). All PCR reactions were performed in a Bio-Rad MyCycler thermocycler. Denaturing Gradient Gel Electrophoresis (DGGE) was performed following Muyzer et al. [16] in a DGGEK-2401 system coupled to a power supply EPS-300 IIV (CBS Scientific ®, USA). PCR amplified 16S rRNA gene fragments from four bacterial species were used as DGGE migration markers. The fragments were obtained with primers 341F-GC/518R from the DNA of the following bacteria (from top to bottom of a DGGE gel): Pseudomonas aeruginosa PAO1, Escherichia coli K12 CECT 433, Paenibacillus sp. DSM 34, Streptomyces caviscabies ATCC 21619. The ImageJ program [23] at NIH (National Institute of Health, USA), was used to evaluate the relative intensity of the distinct bands from ethydium bromide stained gels. The major DGGE bands were excised from the gels, reamplified, purified, and cloned in pCR2.1-TOPO vector from TOPO TA Cloning Kit (Invitrogen). Inserts were reamplified with GC-tailed forward primers and subjected to a second DGGE analysis together with the original environmental sample for clone selection; the selected clones were sequenced at Macrogen® sequencing services (Macrogen Inc., Korea) Sequences were identified using the Blast tool from NCBI. Statistic analyses 4 ANOVA analysis [25] was performed